antiepileptic and antiepileptogenic performance of carisbamate after...

Antiepileptic and Antiepileptogenic Performance ofCarisbamate after Head Injury in the Rat: Blind andRandomized Studies

Clifford L. Eastman, Derek R. Verley, Jason S. Fender, Tessandra H. Stewart, Eytan Nov,Giulia Curia, and Raimondo D’AmbrosioDepartments of Neurological Surgery (C.L.E., D.R.V., J.S.F., E.N., G.C., R.D.) and Neurology and Regional Epilepsy Center(R.D.), Graduate Program in Neurobiology and Behavior (T.H.S.), University of Washington School of Medicine, Seattle,Washington

Received September 17, 2010; accepted November 30, 2010

ABSTRACTCarisbamate (CRS) exhibits broad acute anticonvulsant activityin conventional anticonvulsant screens, genetic models of ab-sence epilepsy and audiogenic seizures, and chronic sponta-neous motor seizures arising after chemoconvulsant-inducedstatus epilepticus. In add-on phase III trials with pharmacore-sistant patients CRS induced �30% average decreases inpartial-onset seizure frequency. We assessed the antiepilepto-genic and antiepileptic performance of subchronic CRS admin-istration on posttraumatic epilepsy (PTE) induced by rostralparasaggital fluid percussion injury (rpFPI), which closely rep-licates human contusive closed head injury. Studies were blindand randomized, and treatment effects were assessed on thebasis of sensitive electrocorticography (ECoG) recordings. An-tiepileptogenic effects were assessed in independent groups ofcontrol and CRS-treated rats, at 1 and 3 months postinjury,

after completion of a 2-week prophylactic treatment initiated 15min after injury. The antiepileptic effects of 1-week CRS treat-ments were assessed in repeated measures experiments at 1and 4 months postinjury. The studies were powered to detect�50 and �40% decreases in epilepsy incidence and frequencyof seizures, respectively. Drug/vehicle treatment, ECoG analy-sis, and [CRS]plasma determination all were performed blind. Wedetected no antiepileptogenic and an equivocal transient anti-epileptic effects of CRS despite [CRS]plasma comparable with orhigher than levels attained in previous preclinical and clinicalstudies. These findings contrast with previous preclinical datademonstrating large efficacy of CRS, but agree with the aver-age effect of CRS seen in clinical trials. The data support theuse of rpFPI-induced PTE in the adolescent rat as a model ofpharmacoresistant epilepsy for preclinical development.

IntroductionCarisbamate [CRS; (S)-2-O-carbamoyl-1-o-chlorophenyl-eth-

anol)] is an investigational neuromodulator that has a broadspectrum of anticonvulsant activity in numerous preclinicaltests (Table 1). In comparison with eight marketed antiepilepticdrugs (AEDs), CRS’s anticonvulsant potency ranked fourth,first, second, and first for seizures evoked in the mouse bymaximal electroshock, pentylenetetrazole, bicuculline, and pi-crotoxin, respectively (Novak et al., 2007). CRS has also dem-onstrated anticonvulsant activity in kindling, lamotrigine-resis-

tant kindled rat, and genetic models of absence epilepsy andaudiogenic seizures and suppresses the chronic spontaneousmotor seizures that arise after chemoconvulsant-induced statusepilepticus (SE) (Novak et al., 2007). Grabenstatter and Dudek(2008) rigorously examined the effects of acute administrationof CRS on chronic spontaneous motor seizures after kainate-induced SE. Seizure frequency was reduced by �75%, and CRSseemed more efficacious than topiramate (Grabenstatter et al.,2005). Similarly encouraging results were obtained in a studywith prolonged lithium-pilocarpine-induced SE in which a1-week course of twice-daily injections of a fixed dose of CRSbegun after the onset of SE resulted in a delay or prevention ofthe appearance of spontaneous motor seizures in the chronicperiod. This has been interpreted as indicative of a possibleantiepileptogenic, or disease-modifying, effect of CRS (Andre etal., 2007). Thus, CRS has demonstrated a broader spectrumof activity in preclinical tests than other marketed AEDs(Rogawski, 2006).

This work was supported by Johnson and Johnson Pharmaceutical Re-search and Development, LLC, and the National Institutes of Health NationalInstitute of Neurological Disorders and Stroke [Grant NS053928] (to R.D.).

Disclosure: In 2006, R.D. served as a paid consultant to Johnson andJohnson.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.110.175133.

ABBREVIATIONS: CRS, carisbamate; AED, antiepileptic drug; ECoG, electrocorticography; G1, grade 1; G2, grade 2; G3, grade 3; PTE,posttraumatic epilepsy; rpFPI, rostral parasagittal fluid percussion injury; SE, status epilepticus.

0022-3565/11/3363-779–790$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 336, No. 3Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 175133/3666020JPET 336:779–790, 2011 Printed in U.S.A.

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However, the translation of encouraging preclinical find-ings to the clinic has been an incomplete success. Althoughthe introduction of numerous AEDs over the past 30 yearshas provided a wider range of therapeutic options and im-proved the tolerability of pharmacotherapy, the proportion ofpatients with poor seizure control has not substantiallychanged (Duncan et al., 2006; French, 2007), and no agenthas been identified to cure epilepsy (Temkin, 2009). Thus,the preclinical investigation of putative AEDs is not idealand could benefit from several refinements. First, more com-prehensive studies could be conducted using chronic sponta-neous seizure models, including acquired epilepsy modelsbased on etiologically realistic insults (Meldrum, 2002;Schmidt and Rogawski, 2002; Stables et al., 2002; White,2002, 2003; Kwan and Brodie, 2003; Loscher and Schmidt,2004; D’Ambrosio and Miller, 2010a,b). Second, the antiepi-leptic assessment could be obtained for prolonged drug expo-sure. The acute effects of AED administration may not ade-quately reflect the performance of drugs administeredchronically in the clinical setting (Loscher , 2007). They alsocannot detect antiepileptic effects that develop slowly, overdays to weeks, as observed with valproate (Loscher andHonack, 1995; Eastman et al., 2010). Third, preclinical stud-ies should probe the entire spectrum of epileptic seizures byelectrocorticography (ECoG) and not be exclusively based onconvulsive behavior. In humans, many epileptic seizures arenot accompanied by overt or distinctive behavioral outputsuch as convulsions, and human nonconvulsive seizures aremore often pharmacoresistant than tonic-clonic convulsions(Juul-Jensen, 1986; Mattson et al., 1996; Semah et al., 1998).Finally, blinded and randomized studies would help ensurean unbiased preclinical workup (Crossley et al., 2008; Philipet al., 2009).

Thus, we have assessed the antiepileptic and antiepilepto-genic effects of subchronically administered CRS on chronicspontaneous seizures in an etiologically realistic model ofpharmacoresistant acquired epilepsy in which chronic spon-taneous seizures were detected and quantified by ECoG.Posttraumatic epilepsy (PTE) was induced by rostral para-saggital fluid percussion injury (rpFPI) in the rat, which ismechanically identical to human contusive head injury and

reproduces many of its pathophysiological sequelae (Thomp-son et al., 2005), including an epileptic syndrome featuringneocortical and limbic seizures (D’Ambrosio et al., 2004,2005, 2009) that are fully blocked by halothane, partiallyresponsive to valproate, but resistant to carbamazepine(Eastman et al., 2010). These studies were conducted in ablind and randomized fashion, using dosing protocols de-signed to maintain blood levels of CRS in a therapeutic rangefor the duration of the on-drug phase of each study. Despitestatistical power adequate to detect at least �40% reductionin seizure frequency and �50% reduction in incidence ofepilepsy, these studies did not demonstrate significant anti-epileptic or antiepileptogenic activity.

Materials and MethodsAnimals. Outbred 32- to 36-day-old male Sprague-Dawley rats

(Charles River Laboratories, Inc., Wilmington, MA) were housed in aspecific-pathogen-free facility two to three per cage to allow social-ization, under controlled conditions of temperature and humidityand a 12-h light/12-h dark cycle. Water and food were available adlibitum. All experimental procedures were approved by the Univer-sity of Washington Institutional Animal Care and Use Committee.

Fluid Percussion Injury. Rostral parasaggital fluid percussioninjury was performed as detailed previously (D’Ambrosio et al., 2009;Eastman et al., 2010) with care to ensure consistent procedure. Inbrief, animals were anesthetized (4% halothane), intubated, andmechanically ventilated (1–1.5% halothane, 30% O2, and air). Coretemperature was maintained at 37°C with a heat pad. A 3-mm burrhole was drilled 2 mm posterior to bregma and 3 mm from themidline over the right convexity. Animals were disconnected fromthe ventilator for injury, and an 8-ms, 3.4- to 3.7-atm pressure pulsewas delivered through the FPI device (Scientific Instruments, Uni-versity of Washington) and measured by a transducer (MeasurementSpecialties, Hampton, VA). To standardize posttraumatic hypoxia,the mechanical ventilation was resumed 10 s after injury. Injuredanimals had righting times in excess of 10 min, and acute mortalityfrom posttraumatic complications was �11%.

Electrode Implantation and Video-ECoG. Epidural electrodeswere implanted as detailed previously (D’Ambrosio et al., 2009). Inbrief, epidural electrodes (stainless-steel screws of � � 1 mm) wereimplanted through guiding craniotomies (� � � 0.75 mm) with careto avoid brain compression or damage to the underlying dura thatsometimes induces artifactual epileptiform discharges. The ECoGmontage consisted of five epidural electrodes: a reference electrodeplaced midline in the frontal bone and two electrodes per parietalbone, placed at coordinates bregma 0 mm and �6.5 mm, 4 mm fromthe midline. All electrodes were connected through insulated wire togold-plated pins in a plastic pedestal, and the entire assembly wascemented onto the skull with dental acrylic (Jet; Lang Dental Man-ufacturing Co., Wheeling, IL). Because of stresses peculiar to theseexperiments initial batches of animals showed a higher incidence ofheadset loosening than in our previous studies, which resulted innoisy recordings over time. Thus, acrylic headsets were more se-curely adhered with VetBond (World Precision Instruments, Inc.,Sarasota, FL) in later batches, which improved their mechanicalresilience and the proportion of useful recordings. Rats were housedseparately after implantation to protect headsets. When possible,lost headsets were reimplanted once. Electrical brain activity wasamplified (�5000) and filtered (0.3-Hz high-pass, 100-Hz low-pass)using a Neurodata 12 or a M15 amplifier (Grass Instruments,Quincy, MA), acquired at 512 Hz per channel, stored, and analyzedon computers equipped with SciWorks and Experimenter V3 soft-ware (Datawave Technologies Inc., Longmont, CO), and DT3010acquisition boards (DataTranslation Inc., Marlboro, MA). One-dayECoG recordings were performed two or three times weekly. Videos

TABLE 1Preclinical evaluation of carisbamate

Test Mouse Rat

Maximal electroshock �a �b

Pentylenetetrazole �a �b

Bicuculline �a �b

Picrotoxin �a �b

Corneal kindling N.D. �b

Hippocampal kindling N.D. �b

Lamotrigine-resistant amygdale kindled rat N.A. �b

6-Hz seizures N.D. �b

Li�-pilocarpine SE N.D. �b

Audiogenic seizure �b �c

Genetic generalized spike-wave seizure(GAERS rat)

N.A. �c

Chronic spontaneous seizure(Li�-pilocarpine SE)

N.D. �d

Chronic spontaneous seizure (kainate SE) N.D. �e

N.D., not determined; N.A., not applicable.a Novak et al., 2007.b White et al., 2006.c Francois et al., 2008.d Andre et al., 2007.e Grabenstatter and Dudek, 2008.

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were recorded using digital cameras connected to videocassette re-corders. Each camera was used to monitor a maximum of two cages(one animal per cage) to permit detection of subtle changes in be-havior.

Quality Control of Surgical Procedures. As in previous work,we took precautions to ensure that chronic epileptiform ECoG eventswere induced by FPI and not by cortical damage caused by substan-dard drilling or by heating or compression during epidural electrodeimplantation. Thus, the skull and drill bit were cooled during drillingwith room-temperature sterile saline, and we took care never todeform the skull or the dura with the drill bit. In addition, the depthof epidural electrodes was carefully measured to avoid brain com-pression (D’Ambrosio et al., 2009).

Identification of Seizures. Random samples of both video andECoG of awake and sleep patterns were first examined in drug-treated animals to ensure CRS did not affect the typical ECoGpatterns of normal rat brain activity and our capability to recognizeseizures. ECoG was then manually scrolled offline as described pre-viously (D’Ambrosio et al., 2004, 2005, 2009). Seizures were charac-terized by epileptiform ECoG patterns with trains of 150- to 250-ms-long spikes, initial amplitudes exceeding twice the previous 2-sbaseline with ictal behavioral changes as described previously. Theseseizures are seen only in FPI and not in sham-injured animals. Consis-tent with our previous demonstration of the existence of short clinicalseizures in posttraumatic epileptic rat and humans (D’Ambrosio et al.,2009), observed seizure duration ranged from 1 s to 10 min. Seizuresoccurring within 5 s of each other were defined as one seizure event.Because all FPI animals were at risk of developing PTE, we diagnosedepilepsy upon their first proven ictal electroclinical event (Beghi et al.,2005; Fisher et al., 2005; Fisher and Leppik, 2008). ECoG seizures werecategorized as: 1) grade 1 (G1), if appearing to be limited to a corticalfocus; 2) grade 2 (G2), when appearing first in a limited cortical areaand then spread to other cortical areas; and 3) grade 3 (G3), if appearingbilateral at their cortical onset. G1 and G2 seizures are generated by aneocortical focus, whereas G3 events have a likely limbic origin(D’Ambrosio et al., 2004, 2005, 2009). In accord with previous work(D’Ambrosio et al., 2004, 2005), age-dependent idiopathic seizures, typ-ically 1- to 10 s long, bilateral in onset, with a sharp-wave pattern thatwas larger in amplitude in the parietal-occipital cortex, were not seen inthis study because they typically appear after �5 to 6 months of age.

Plasma CRS Determination. Blood (0.3–0.5 ml) for plasma CRSdetermination was withdrawn from the tail vein into a microtainertube with K2EDTA (BD Biosciences, San Jose, CA). Blood sampleswere spun at 3000g for 10 min in a Marathon 6K tabletop centrifuge(Thermo Fisher Scientific, Waltham, MA). Plasma supernatant wasseparated, frozen, coded for blind analysis, and shipped on dry ice toKeystone Analytical (North Wales, PA), which measured CRS levelsby high-performance liquid chromatography.

Carisbamate Dosing. Pilot studies were conducted to identifyoral dosing regimens capable of stably maintaining CRS plasmalevels. For this purpose, naive rats age- and gender-matched to ourexperimental animals were administered CRS (Johnson and John-son Pharmaceutical Research and Development, Raritan, NJ) sus-pended in a condensed milk-based vehicle. Early studies showed asteady decline from targeted steady-state plasma levels during ad-ministration of 90 mg/kg CRS three times daily (Fig. 1A), presum-ably because of auto-induction of its metabolism. However, [CRS-]plasma showed no similar decline from target levels in ratsadministered increasing doses of CRS (Fig. 1B). The first 7 days ofthis regimen were used for the antiepileptic study, whereas theantiepileptogenesis study used a modified regimen (Table 2) thatincluded a higher dose in the first days of treatment to boost possibleantiepileptogenesis (Fig. 1B). All oral doses of CRS were delivered bysyringe in a volume of �0.2 ml in 75% Borden’s Eagle Brand sweet-ened condensed milk in sterile water, a vehicle in which control anddrug preparations were visually indistinguishable. For intraperito-neal injection, CRS was solubilized in 10% Solutol (generously pro-vided by BASF, Ludwigshafen, Germany) according to the manufac-turer’s instructions.

Antiepileptic Assay. The study protocol is shown in Fig. 2C.Because different seizure types develop with different time coursesafter rpFPI (D’Ambrosio et al., 2004, 2005), antiepileptic testing was

Fig. 1. Pilot carisbamate pharmacoki-netic studies. A, steady-state plasma CRSlevels decline during administration of afixed dose of 90 mg/kg three times daily(n � 9–10 per time point). B, the declinein steady-state plasma CRS levels is pre-vented by administration of increasingdoses of CRS three times daily as shown(n � 3–10 per time point). In both studies,doses were delivered at 7 AM, 3 PM, and11 PM daily.

TABLE 2Dosing protocols for the antiepileptic and antiepileptogenic studies

DayCRS Dosea

Antiepileptic Studyb Antiepileptogenesis Studyc

mg/kg1 45, 70 80 60d, 1002 80 1073 90 1144 95 1225 105 1296 115 1367 130 1438 1519 158

10 16511 16512 16513 16514 165

a CRS was administered orally each day at 7AM, 3 PM, and 11 PM at theindicated doses.

b CRS administration began on postinjury week 5 and/or 17.c CRS administration began 15 min after FPI.d Administered intraperitoneally.

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conducted at 4 to 6 weeks and 16 to 18 weeks after rpFPI (Fig. 2C) toassess the efficacy of CRS in controlling G1 seizures and G2 � G3seizures, respectively. Assigned treatments were administered onpostinjury weeks 5 or 17. CRS was administered in increasing dosesaccording to the schedule determined by the pilot pharmacokineticstudy (Table 2). To ensure adequate and stable blood levels of CRSduring on-drug data acquisition, video-ECoG recordings commencedafter a 2-day wash-on period. Blood samples were collected at least12 h before the first week 5 on-drug ECoG recording and at the endof a 2-day washoff period (week 6). On-drug blood samples werecollected at most 2 h before CRS administration to sample troughlevels of CRS. Each animal served as its own control. The effect ofCRS on neocortical seizures was assessed 5 weeks after injury in twogroups of 10 animals that all met a three seizures per baseline weekcriterion (Eastman et al., 2010) on postinjury week 4. The effect ofCRS on limbic (G3) and spreading neocortical (G2) seizures wasassessed in a cohort of seven rats, three of which had been used totest the antiepileptic effects of CRS at 4 to 6 weeks. Thus fouranimals received CRS on week 17 only. Because kindling in thepresence of CRS did not induce CRS resistance (Klein et al., 2007), itis unlikely that week 5 CRS exposure affected CRS responsiveness atweek 17.

Antiepileptogenesis Assay. The study protocol is shown in Fig.2A. CRS or vehicle dosing was as in Table 2. Blood for [CRS]plasma

determination was drawn from all animals 15 min to 2 h before theafternoon dose on the second and ninth day after the start of CRSadministration. Measured [CRS]plasma thus approximate trough lev-els of the drug.

Blinding Measures. Antiepileptic and antiepileptogenesis stud-ies both were fully masked. Rats were randomized to treatment withdrug or vehicle before the first ECoG recordings (antiepileptic) or thestart of drug treatment (antiepileptogenesis). Treatment suspen-sions of vehicle or drug were supplied in visually indistinguishablevials labeled only with coded identifiers. Thus, treatments wereadministered by investigators who were blind to both the contents oftreatment suspensions and the group assignments of individual sub-jects. All ECoG data were analyzed by investigators who wereblind to the subject, treatment group, or collection date of datafiles. The decision to discard a noisy ECoG file was made blind tofile identity. Data file identifiers remained hidden until all datahad been collected and verified. Blood samples were collected andcoded, and CRS levels were measured blind to subject, treatment,and dosing protocol.

Fig. 2. Study design and power analyses. A, antiepileptogenesis study. Immediately after rpFPI, animals were randomly assigned to treatment witheither vehicle or CRS with equal probability. Treatment commenced 15 min after injury and continued for 2 weeks. Epidural electrodes were implanted2 weeks after injury, and 48 h of ECoG recordings were obtained from each rat on postinjury weeks 4 and 12. B, nonparametric power analyses forantiepileptogenesis study. Graph shows group sizes required to provide 80% power to detect the indicated antiepileptic effects (one-tailed � � 0.05).Œ indicate group sizes required to detect uniform decreases in seizure frequency. E indicate group sizes required to detect increases in the incidenceof seizure-freedom. C, antiepileptic study. Rats received rpFPI, and epidural electrodes were implanted 3 weeks after injury, allowing 1 week forrecovery before predrug recording. Animals were randomized to treatment groups in a 1:1 proportion before their first ECoG recordings. Each 3-weektesting sequence consisted of three 1-day ECoG recordings on the first week (Baseline I) before drug exposure, on the second week of testing (Drugon) during which the animal was on the drug after a 48-h wash-on period, and on the third week (Baseline II) during which the animal was off thedrug after the washoff period. Thus, 72 h of ECoG recordings were acquired from each rat during each week of the 3-week test sequence (�216 h totalper rat). D, nonparametric power analysis for antiepileptic study. Graph shows group size required to provide 80% power to detect uniform decreasesin seizure frequency (one-tailed � � 0.05). Data are from Eastman et al. (2010).

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Data Analysis. In all experiments, video-ECoG data were ana-lyzed off-line, in random sequence, by trained investigators. Seizuresof grades 1, 2, and 3 were identified, and seizure frequencies (events/h), mean seizure durations (s), and times spent seizing (s/h), weredetermined. To facilitate comparison with previous studies in whichseizures were arbitrarily defined based on duration, parallel analy-ses of all seizures versus seizures lasting �5 s are presented. Seizurefrequencies and times spent seizing were logarithmically trans-formed before comparison using t tests.

Nonparametric power analyses have shown this strategy to bemore sensitive to detect antiepileptic effects of investigational drugsand robust to the confounding effect of nonresponders (Eastman etal., 2010). Because the logarithm of zero is � and the occurrence ofno seizures in a finite observation period only suggests a seizurefrequency of less than one per observation period (72 h per time pointfor the antiepileptic study and 48 h per time point for the antiepi-leptogenic study), observations of zero seizure frequency or timeseizing were assigned conservative floor values (Eastman et al.,2010) of 1/72 and 1/48 in the antiepileptic and antiepileptogenicstudies, respectively. For determination of the antiepileptic effect ofCRS, decreases from predrug (week 4 or 16) levels of seizure fre-quency and time seizing were evaluated for the corresponding on-drug (weeks 5 or 17) and posttreatment (weeks 6 or 18) periods usingpaired t tests (Eastman et al., 2010). Paired decreases in seizureduration were evaluated using Wilcoxon’s matched pairs signedranks test. Antiepileptic effects on seizure frequency or time seizingshown in Figs. 4 and 6 were computed as mean differences betweenlog-transformed pretreatment and posttreatment frequencies ortimes seizing (e.g., effect � log(frequencyt1/frequencyt2), in which t1is pretreatment baseline, and t2 is the week either on treatment orpost-treatment). We have shown previously (Eastman et al., 2010)that optimal analysis of seizure frequency data from FPI rats (pairedt test using logarithmically transformed data) of a group of just eightrats is sufficient to provide at least 80% power to detect a drug-induced �45% decrease in seizure frequency in the absence of non-responders. In the antiepileptogenesis study, seizure frequencies(events/h), mean seizure durations (s), and times spent seizing (s/h)were determined 4 and 12 weeks after rpFPI. These data werecompared between groups of rats that had been treated with vehicleor CRS. Antiepileptogenesis was assessed at postinjury weeks 4 and 12on the basis of unpaired comparisons of vehicle- and CRS-treated log-transformed seizure frequency and time spent seizing (unpaired t test).Untransformed seizure durations were evaluated using the Mann–Whitney test. Because logarithms of values less than one are negative,log-transformed frequencies and times seizing were plotted as log(fre-quency) � log(floor) such that increases in frequency are represented aspositive deflections from a zero floor. Treatment randomizations werecarried out with the aid of Excel 2002 (Microsoft Inc., Redmond, WA)and Statistics101 (http://www.statistics101.net) resampling statisticssoftware. Data were compiled and analyzed in Excel 2002, and statis-tical analyses were conducted using SPSS (version 10; SPSS Inc., Chi-cago, IL). In keeping with our focus on identifying antiepileptic orantiepileptogenic activity, statistical tests were one-tailed. All resultsare presented as mean S.E.M.

Nonparametric Power Analysis. To estimate the experimentalgroup size required to detect reductions in seizure frequency in theantiepileptogenesis study, a nonparametric power analysis was con-ducted as described previously (Eastman et al., 2010). In brief, twosamples of size N were randomly drawn from a database containingseizure frequencies previously measured at 4 weeks postinjury fromidentically injured rats. Treatment was simulated in one sample byuniformly reducing the sampled frequencies by a set percentage(effect size), and both samples were logarithmically transformed andcompared using a one-tailed unpaired Welch’s t test (� � 0.05). Thisoperation was repeated 105 times for each specified N and effect size,correct detections of the specified treatment effects were counted,and the statistical power was computed as proportion of the 105

comparisons yielding correct (statistically significant) detections of

the simulated treatment effects. The seizure frequency databaseincluded 102 frequencies (one per animal) obtained from injured ratsthat were recorded for 48 h 4 weeks after injury. In 23 of these rats,no seizures were observed in the 48 h of recording. The mean seizurefrequency (n � 102) was 1.9 0.4 events/h, and the median was 0.34events/h.

ResultsAntiepileptogenesis Study. At 4 weeks postinjury, 2

weeks after cessation of CRS treatment, there was no detect-able prophylactic effect on FPI-induced PTE in 22 control and27 CRS rats (Fig. 3). The incidence of epilepsy was 82 and85% and the frequency of seizures was 1.4 0.6 and 1.5 0.4seizures/h in vehicle- and CRS-treated animals, respectively.Both groups are consistent with our historical data(D’Ambrosio et al., 2004, 2005). The mean log-transformedfrequencies of (Fig. 3A) and times spent in (Fig. 3E) G1, G2,G1 � G2 (neocortical), G2 � G3 (spreading), and G1 � G2 �G3 (all) seizures in CRS-treated animals all were nominallyelevated with respect to controls, and no seizure type wassignificantly decreased by CRS treatment (all p � 0.35, un-paired t test). The mean duration of seizures (Fig. 3C) alsowas not decreased significantly in the CRS-treated group(Mann–Whitney test; all p � 0.3). At 12 weeks postinjury, 10weeks after cessation of CRS treatment, the log-transformedfrequency (Fig. 3B), time spent seizing (Fig. 3F), and theduration (Fig. 3D) still did not differ between control (n � 13)and CRS-treated (n � 17) animals (all p � 0.25; unpaired ttest). The mean duration of seizure was also undiminished inthe CRS-treated group (all p � 0.2; Mann–Whitney).

In CRS-treated rats, mean plasma CRS (Fig. 3G) was 29 6 �g/ml 2 days after the start of treatment and 11 2 �g/mlon the ninth day of treatment. In vehicle-treated rats, plasmaCRS was 0.24 0.10 �g/ml on day 2 of the treatment periodand increased to 1.90 0.40 �g/ml on day 9. The detection ofCRS in untreated controls was unexpected and was traced tothe fact that, in contrast to the pilot study, CRS-treatedanimals were caged together with controls. The CRS-treatedanimals created a fecal source of CRS that was consumed bycontrols. There was no indication of a relationship betweenthe [CRS]plasma measured during prophylactic treatment andthe frequency of seizure at either 4 or 12 weeks postinjury(Fig. 3H). The highest log-transformed seizure frequencies(�0.5 3.16 events/h) were observed in control and CRS ratsthat had mean [CRS]plasma ranging from 0.7 to 46 �g/mlduring prophylactic treatment. There was also no significantcorrelation between plasma CRS and time spent seizing (notshown). It is therefore unlikely that an antiepileptogeniceffect of CRS would have been observed at a higher dose.

Antiepileptic Study. The antiepileptic efficacy of CRSwas first assessed at 4 to 6 weeks postinjury, when seizurespredominantly originate from a perilesional neocortical focusand most of them do not spread (G1). Thus, limbic seizures(G3) and spreading neocortical seizures (G2) were rare com-pared with nonspreading neocortical seizures (G1; Fig. 4, Cand D). As reported previously (Eastman et al., 2010), seizurefrequencies varied widely among the individual rats in eachtreatment group (Fig. 4, A and B). The mean pretreatmentbaseline frequencies of seizure were comparable in controlversus CRS group after logarithmic transformation (�0.01 0.16 in controls; 0.03 0.24 in CRS group). Also the pretreat-


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ment duration of seizure was comparable in the two groups(6.7 1.1 s in controls; 5.8 0.9 s in the CRS group). Themean log-transformed frequency of seizure (all types) did notdecrease during week 5 treatment in either treatment group,and the nominal decrease, with respect to week 4, observed inCRS-treated rats in the week after cessation of treatment

(Fig. 4G) did not reach significance (paired t test; p � 0.10).We also detected no significant decreases in the mean log-transformed frequency of any type of seizure either during(all p � 0.3; paired t test) or after (all p � 0.1; paired t test)week 5 CRS treatment (Fig. 4D). Likewise, no significanttime- or treatment-related decreases were detected in the

Fig. 3. CRS did not decrease FPI-induced epileptogenesis. A, log-trans-formed frequencies of G1, G2, G3, neo-cortical (G1 � G2), spreading (G2 �G3), and all (G1–3) seizures in vehicle-treated (CON) and CRS-treated rats at4 weeks post-FPI. B, log-transformedfrequencies of seizure, as in A at 12weeks postinjury. C and D, duration ofseizures at 4 (C) and 12 (D) weeks afterinjury. E and F, log-transformed timesspent seizing at 4 (E) and 12 (F) weekspost-FPI. Both vehicle- and CRS-treated groups had seizure frequencyand duration and time spent seizingconsistent with historical data fromuntreated animals. G, the mean [CRS-]plasma remained above 10 �g/mlthrough day 9 of the 14-day protocol.The low, but detectable, levels of CRSseen in some vehicle-treated (CON) ratswere attributed to coprophagous inges-tion of CRS excreted by CRS-treatedcage mates. H, despite the wide range of[CRS]plasma measured in individualrats, there was no apparent relation-ship between [CRS]plasma and seizurefrequency at either 4 or 12 weeks afterinjury. Plasma CRS levels are the meansof measurements at days 2 and 9.

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duration of all seizures (all p � 0.25; Wilcoxon) in eithervehicle- or CRS-treated rats (Fig. 4H). Log-transformedtimes spent seizing were nominally elevated during week 5treatment in both vehicle and CRS groups, and the post-treatment decreases in time seizing did not approach signif-icance (both p � 0.25; paired t test) in either group (notshown).

Although CRS did not significantly affect G1 or G2 neocor-tical seizures, there was limited evidence pointing to a pos-sible effect on the much rarer G3 events that originate froma subcortical focus. There were no significant time-dependent(Fig. 4C) or treatment-dependent (Fig. 4D) decreases in log-transformed frequency of G3 seizures (p � 0.3; paired t test),but log-transformed times spent seizing seemed to be signif-

icantly decreased during (week 5) and after (week 6) CRStreatment (both p � 0.047; Fig. 4F). It is worth noting,however, that only 6/10 rats exhibited G3 seizures at 1 monthpostinjury, and G3 frequency was very low in those that did:0.027 0.005 events/h versus 2.2 0.7 events/h and 0.19 0.07 events/h for seizures of grades 1 and 2, respectively inthe 20 (control � CRS) rats recorded at week 4 postinjury.Thus, this study was not designed to accurately and confi-dently assess decreases in the frequency of G3 seizures.

CRS levels in plasma samples obtained �48 h after start oftreatment and before week 5 recordings were 14.80 3.7�g/ml (range 6.40–28.30 �g/ml). Data from CRS-treated an-imals revealed no significant correlation (rho � 0.45; p �0.26; Spearman) between [CRS]plasma and the observed anti-

Fig. 4. CRS had no effect on neocortical seizures at 5 weeks post-FPI. A and B, frequencies of all seizures recorded from individual vehicle-treated (A)and CRS-treated (B) rats before, during, and after week 5 treatment are shown on logarithmic scale. C and D, log-transformed frequencies of G1, G2,and G3 seizures in the vehicle-treated (C) and CRS-treated (D) groups. There was no significant decrease in any seizure type. E and F, log-transformedtimes spent in G1, G2, and G3 seizure in the vehicle-treated (E) and CRS-treated (F) group. There was no significant decrease in the neocorticalseizures that predominate at this stage of the evolution of FPI-induced PTE. Decreases, with respect to week 4, in the times spent in rare G3 seizuresseemed significant at both weeks 5 and 6 (�, p � 0.047). G, log-transformed frequency of all seizures in the vehicle- and CRS-treated groups. Thefrequency of seizure was not significantly decreased with respect to the week 4 pretreatment baseline at any time point in either group. H,log-transformed seizure duration was stable in both treatment groups over the course of the study. I, the antiepileptic effects determined during (week5) and after (week 6) CRS treatment showed no significant correlation with plasma CRS levels. Effect is computed as log(frequencyweek 4/frequencyweek x).


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epileptic effect of CRS as indicated by week 4 minus week 5difference in log-transformed seizure frequencies (Fig. 4I).CRS effects at week 6 were also not significantly correlatedwith [CRS]plasma (rho � 0.21; p � 0.63; Spearman). Thus,there is no evidence that higher plasma CRS levels may havebeen more effective.

To better examine the possibility that CRS may particu-larly affect limbic seizures, a cohort of seven animals waschallenged with CRS at 16 to 18 weeks post-FPI. At that timespreading seizures (G2 and G3) predominated (Fig. 5, C-F)and limbic seizures (G3) accounted for 30 to 40% of observedseizures. The mean log-transformed frequency of and timespent seizing in G3 seizures were nominally elevated overpredrug levels both during (week 17) and after (week 18) CRStreatment (Fig. 5, C and D), and there was no decreases inoverall seizure frequency or in the frequency of any type ofseizure (all p � 0.33).

Evaluation of CRS Effects on Seizures of DifferentDurations. To facilitate comparison with previous studiesthat define seizures as lasting at least 5 s, the data presentedabove were reanalyzed after breaking down the seizures inclasses based on their duration: �1 to �2 s, �2 to �5 s, �5to �15 s, �15 to �30 s, and �30 s. The resulting seizureduration histograms indicate no antiepileptic effect of CRSon seizures of any duration either at 5 weeks (Fig. 6A) or 17weeks (Fig. 6B) postinjury and no antiepileptogenic effect ofthe prophylactic CRS treatment at either 4 weeks (Fig. 6E) or

12 weeks (Fig. 6F) post-FPI. Exclusion of the shorter (1–5 s)clinical seizures resulted in a 67 2% decrease in the ap-parent frequency of seizure, a 107 6% increase in theapparent duration of seizure, and a 41 2% decrease in timespent seizing, but led to similar inferences regarding both theantiepileptic (Fig. 6, C and D) and antiepileptogenic (Fig. 6, Gand H) activities of CRS.

DiscussionWe report the first assessment of the effects of CRS on

neocortical and limbic partial seizures induced by head in-jury in the rat. The study incorporates several strategiesintended to facilitate translation to the clinical setting: 1) theepileptogenic insult is etiologically realistic to better capturemechanisms operating in human PTE, 2) ECoG is used tosensitively detect and comprehensively examine the epilepticsyndromes; 3) the antiepileptic effect on spontaneous sei-zures is evaluated during a subchronic treatment that main-tains stable blood levels of the drug, to better simulate theclinical conditions under which AEDs have to perform, and4) the study is conducted in a blind and randomized fashion.The main findings of this study, that CRS demonstrated nomeaningful antiepileptogenic or antiepileptic effects on FPI-induced PTE, are in contrast to previous preclinical studiesperformed with different models and convulsive endpoints.

Fig. 5. CRS decreased neither neocortical nor limbic seizures at 17 weeks postinjury. A and B, frequencies (A) and durations (B) of all seizures recordedfrom individual rats before, during, and after week 17 CRS administration are shown on a logarithmic scale. C and D, mean log-transformedfrequencies of (C) and times spent in (D) G1, G2, and G3 seizures before, during, and after week 17 treatment with CRS. There was no significantdecrease in any seizure type. E and F, mean log-transformed frequencies of (E) and times spent in (F) neocortical (G1 � G2) and limbic (G3) seizuresbefore, during, and after week 17 treatment with CRS. There was no significant decrease in either seizure class.

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Examples of seizures observed during CRS treatment areshown in Fig. 7.

Lack of Antiepileptogenic Effect of Carisbamate af-ter FPI. Although a possible antiepileptogenic activity hasbeen inferred based on the efficacy of CRS in kindlingmodels (Novak et al., 2007) and a report that the emer-gence of chronic spontaneous motor seizures after lithium-pilocarpine-induced status epilepticus was delayed afterprophylactic CRS treatment (Andre et al., 2007), we foundno evidence for an antiepileptogenic effect of CRS afterhead injury. Mean log-transformed seizure frequencies andtimes seizing all were nominally higher in CRS-treated rats

than in controls at both 1 and 3 months postinjury (Fig. 3).The lack of correlation between [CRS]plasma and seizure fre-quency in individual subjects (Fig. 3H) provides additionalevidence that inadequate dosing does not account for theapparent inactivity. The [CRS]plasma maintained for the firstweek of prophylactic treatment in this study was probablyhigher than that attained (but not measured) in a studyreporting CRS to have an antiepileptogenic effect after aweeklong treatment begun during pilocarpine-induced SE(Andre et al., 2007). Given the steady decline in [CRS]plasma

achieved during repeated administration of CRS at a fixeddose (Fig. 1A) and the good absorption of orally administered

Fig. 6. Antiepileptic and antiepilepto-genic properties of CRS on seizures ofdifferent duration. A and B, histogramsshowing the frequency of seizures of dif-fering durations before, during, and af-ter CRS treatment at 4, 5, and 6 weekspost-FPI (A) and at 16, 17, and 18 weeks(B). C and D, antiepileptic effects basedon all detectable clinical seizures (C) orusing only clinical seizures lasting lon-ger than 5 s (D). In either case, no sig-nificant antiepileptic effect was de-tected. E and F, histograms showing thefrequency of seizures of differing dura-tions in vehicle-treated (CON) and CRS-treated rats at 4 (E) and 12 (F) weeksafter injury in the antiepileptogenicstudy. Seizure frequency was not af-fected by prophylactic treatment, andboth the shortest and longest seizuresappeared equally after vehicle or CRS.G and H, log-transformed seizure fre-quencies determined on the basis of alldetectable clinical seizures (G) or justthose lasting longer than 5 s (H) at 4and 12 weeks postinjury in the antiepi-leptogenic study. To display log-trans-formed frequencies as positive valueswith a zero minimum, they are plottedas log(frequencyobserved/frequencyfloor).


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CRS (Mamidi et al., 2007), our extended (2 week) regimen ofescalating doses (160–495 mg/kg per day) that reached 430mg/kg per day by day 7 probably produced higher [CRS]plasma

for a longer period than the 7 days of twice-daily 120 mg/kgintraperitoneal dose that delayed or prevented chronic sei-zures after pilocarpine. Although it cannot be ruled out thatthe apparent antiepileptogenic activity of CRS administeredbefore or during pilocarpine induction of status epilepticusmay reflect an antiepileptic effect on the status epilepticusthat serves as an epileptogenic insult in that model, thediscrepant results may also reflect significant differences inthe mechanisms of epileptogenesis induced by head injuryversus pilocarpine-induced SE.

Lack of Substantial Antiepileptic Effects of Caris-bamate on FPI-Induced Epilepsy. Previous studies haveshown CRS to have potent antiepileptic activity in geneticmodels of audiogenic and absence epilepsies and on the spon-taneous motor seizures that arise after kainate-evoked SE.CRS, at doses of just 20 and 30 mg/kg i.p., completely sup-

pressed sound-evoked seizures in Wistar AS rats, and classicspike and wave discharges in GAERS rats could be com-pletely suppressed after administration of 60 mg/kg CRS(Francois et al., 2008). Using a kainate model of acquiredepilepsy, Grabenstatter and Dudek (2008) reported that thefrequency of spontaneous convulsive seizures in epileptic ratsdecreased by �75% during 6-h monitoring periods after acuteCRS administration. These results were obtained with CRSdoses producing �11�g/ml CRS in plasma at the midpoint ofeach acute monitoring period.

In contrast, CRS maintained at comparable or greater[CRS]plasma for 1 full week demonstrated no convincing an-tiepileptic activity in FPI-induced PTE. Neither the neocor-tical nor the overall frequency of seizure and time spentseizing were significantly diminished by CRS at 1 month(Fig. 4, G and H) or 3 months (Fig. 5, D and F) postinjury. Theobserved effect on limbic seizures (Fig. 4F) may be unreliablebecause of their rarity at this time point (D’Ambrosio et al.,2004, 2005). The effect is also time-limited, because CRS

Fig. 7. Electrographic and behavioral features of G1, G2, and G3 seizures recorded during CRS administration. A, a typical G3 seizure. Activeexploration is evident in frames captured before the onset of electrographic activity that is first detected simultaneously by perilesional (4–5) andcontralateral (1–5) electrodes. Static posture is evident in two frames captured during the electrographic discharge, and movement resumes aftertermination of the G3 seizure. B, a typical G2 seizure. Postural changes are evident in frames taken before onset of an electrographic discharge thatis first detected by the perilesional electrode (4–5) and spreads both caudally and contralaterally to be detected by all four epidural electrodes. Animal’sposture is static in frames captured during the electrographic discharge and movement resumes after termination of the G2 seizure. A head-shakingartifact detected by all four electrodes is clearly distinguishable. C, a typical short G1 seizure. Active exploratory behavior is evident in changes in theanimal’s position in frames captured shortly before the onset of a brief G1 seizure detected only by the perilesional electrode (4–5). Ictal motion arrestis indicated by the animal’s static posture in frames captured (arrest) during the electrographic discharge. Resumption of exploration is evident aftertermination of the discharge. Electrode montage in A applies to A to C. Numbers shown to the left of ECoG traces indicate recording and referenceelectrodes as shown in the Inset. In A to C, dotted boxes show electrographic activity during motion arrest on an expanded time scale. Gray arrowsindicate ECoG seizure onset.

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administered at 17 weeks did not control them (Fig. 5E).Thus, it would probably be of little clinical significance.

Differences from Previous Preclinical Studies. Thesedata show FPI-induced PTE to be resistant to CRS. Ourresults differ starkly from previous preclinical studies, in-cluding those focused on spontaneous seizures. This disparityis not attributable to differences in dosing, statistical power,or seizure detection. The [CRS]plasma maintained in the pres-ent studies were comparable with, or greater than, thoseachieved in preclinical studies demonstrating robust antiepi-leptic or antiepileptogenic effects (Rogawski, 2006) and inclinical trials (Sperling et al., 2010). Power analyses (Fig. 2,B and D) indicate the study designs to be adequate to detecteffects comparable with the antiepileptogenic 45% incidenceof seizure freedom reported by Andre et al. (2007) in thepilocarpine model and substantially smaller than the anti-epileptic �75% decrease in frequency of motor seizure re-ported by Grabenstatter et al. (2008) in the kainate model.The poor performance of CRS in rpFPI-PTE is also not at-tributable to the inclusion, in our analysis, of short (�5 s)clinical seizures that have been ignored in previous studies(D’Ambrosio et al., 2009; D’Ambrosio and Miller, 2010b;Eastman et al., 2010) because longer seizures are not moreresponsive to CRS (Fig. 6).

Thus, the disparities between this and previous preclinicalwork must depend on other features that distinguish thisexperimental approach (Eastman et al., 2010), which we nowrefine to include blind and randomized experimental design.First, the prolonged (1 week) continuous AED exposure usedin our studies are more likely to be affected by the time-dependent functional or metabolic tolerance that occur dur-ing prolonged exposure to therapeutic levels of AEDs(Loscher, 2007) than the acute (hours) exposures used inprevious preclinical studies. Second, previous work has fo-cused exclusively on behaviorally observed convulsive sei-zures, whereas we monitored all seizures, and there is littlereason to expect that etiologically and phenotypically distinctseizures should be similarly responsive to treatment. Humancomplex partial seizures, which are characterized by alteredcognition and motor output depending on regions of onsetand spread and are often nonconvulsive, represent a muchgreater challenge for pharmacological treatment than tonic-clonic convulsions (Juul-Jensen, 1986; Mattson et al., 1996;Semah et al., 1998). The relationship between etiology andpharmacoresponsiveness is not well explored, but acquiredepilepsies tend to be pharmacoresistant (Semah et al., 1998;Callaghan et al., 2007; Hitiris et al., 2007), and human PTE,in particular, seemed more resistant to treatment in a clini-cal trial in which outcomes were evaluated by etiology (Lu etal., 2009). Thus, our study of acquired and predominantlynonconvulsive clinical seizures may contribute to the overallresistance to CRS.

rpFPI in Adolescent Rat as Model of Pharmacoresis-tant PTE. Chronic recurrent spontaneous seizures inducedby rpFPI in the adolescent rat are fully blocked by halothanein all animals and by valproate in those animals with fewand/or exclusively nonspreading seizures. Carbamazepine, incontrast, is ineffective in all animals tested (Eastman et al.,2010). The poor efficacy of CRS we now report further dem-onstrates that rpFPI induces a pharmacoresistant PTE inmost animals. Although the mechanisms of this pharmaco-resistance remain to be elucidated, the realistic contusive

closed head injury and ensuing PTE syndrome probably re-cruit mechanisms similar to those operating in human phar-macoresistant PTE. This model’s record of pharmacoresis-tance is unique and contrasts with the broad responsivenessto virtually all AEDs of chronic seizures induced in SE mod-els. Indeed, with the exception of ethosuximide, all AEDstested have been found to have large effects against sponta-neous motor seizures after pilocarpine, kainate, or amygda-lar stimulation-induced SE (Glien et al., 2002; Loscher, 2002;Grabenstatter et al., 2005; Grabenstatter and Dudek, 2008).

Despite its disparity with previous preclinical work, theequivocal antiepileptic effect of CRS we found in rpFPI-PTEwas not inconsistent with that observed in populations ofpartial-onset pharmacoresistant epilepsy patients in add-onclinical trials (Faught et al., 2008; Sperling et al., 2010),because large effects of CRS were not seen in either case. Noclinical data are available to assess the performance of CRSon patients with pharmacoresistant epilepsy caused by headtrauma, and neither our study nor the add-on clinical trials ofCRS in pharmacoresistant epilepsies may reflect the perfor-mance of CRS on pharmacoresponsive epilepsy patients. Itremains possible that CRS may exhibit exemplary perfor-mance in clinical trials conducted with newly diagnosed ep-ilepsy patients suffering from tonic-clonic convulsions, assuggested by the bulk of previous preclinical work.

ConclusionsFPI-induced PTE in the adolescent rat is resistant to car-

bamazepine, but can be fully blocked by halothane in allanimals and by valproate in some animals (Eastman et al.,2010). The failure of CRS to prevent or reduce epilepticseizures in this study further supports rpFPI-PTE as a modelof pharmacoresistant PTE. In spite of very promising preclin-ical studies using other animal models, clinical trials showedCRS had limited efficacy as add-on treatment for most phar-macoresistant patients. Thus, FPI-PTE may prove useful foridentifying agents effective in the �30% of epilepsy patientswho are now pharmacoresistant.

Acknowledgments

We thank Drs. Steve White and Brian Klein for helpful discussionand service as liaisons with Johnson and Johnson PharmaceuticalResearch and Development, LLC, and Dr. Nancy Temkin for helpfuldiscussion and statistical consultation.

Authorship Contributions

Participated in research design: Eastman, Verley, Fender, andD’Ambrosio.

Conducted experiments: Eastman, Verley, Fender, Stewart, Nov,Curia, and D’Ambrosio.

Performed data analysis: Eastman, Verley, Fender, Curia, andD’Ambrosio.

Wrote or contributed to the writing of the manuscript: Eastmanand D’Ambrosio.

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Address correspondence to: Raimondo D’Ambrosio, Harborview MedicalCenter, Department of Neurosurgery, University of Washington, Box 359915,325 Ninth Ave., Seattle, WA 98104. E-mail: [email protected]

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